Microbiological production of carotene in a medium comprising kerosene



llnited States Patent MICROBIOLOGICAL PRODUCTION OF CARO- TENE IN AMEDIUM COMPRISING KEROSENE Alex Ciegler, Harlow H. Hall, and George E.N. Nelson,

Peoria, Ill., assignors to the United States of America as representedby the Secretary of Agriculture No Drawing. Filed Oct. 19, 1960, Ser.No. 63,697

2 Claims. (Cl. 195-28) (Granted under Title 35, U.S. Code (1952), see.266) A nonexclusive, irrevocable, royalty-free license in the inventionherein described, throughout the world for all purposes of the UnitedStates Government, with the power to grant sublicenses for suchpurposes, is hereby granted to the Government of the United States ofAmerica.

This invention pertains to an improvement in the process of producingbeta-carotene by submerged aerobic fermentation of Blakeslea trispora.

More specifically, this invention relates to the process of producingbeta-carotene by submarged aerobic fermentation of a Blakeslea trisporainoculum in a medium which is markedly improved by the addition of asmall proportion of a commercial kerosene-type petroleum fractionhydrocarbon thereto.

As shown in Anderson, U.S. 2,890,989, it is known to produce fairly goodyields of beta-carotene by the submerged aerobic fermentation ofBlakeslea trispora in an aqueous nutrient medium comprising anassimilable carbon and nitrogen source such as grains, a thiamin source,mineral salts (tap water), a natural fat or oil, a surfaceactive agent,and beta-ionone which latter is added 48 hours after starting thefermentation.

In an effort to further improve the yields of beta-carotene in the abovefermentation process, we have now discovered that by adding a smallproportion of a kerosenetype petroleum fraction hydrocarbon to theculture medium at Zero to 48 hours after starting the fermentation, theproduction of beta-carotene is greatly increased.

The principal object of this invention, accordingly, is an improvementin the fermentation medium so that the production of beta-carotenetherein is greatly increased.

As will be apparent from the results shown in the examples, we have nowdiscovered that the addition of about percent of a kerosene-typepetroleum fraction hydrocarbon initially or after 24 or 48 hours to afermentation of Blakeslea trispora greatly augments the production ofbeta-carotene therein.

EXAMPLE 1 A medium having the following composition was prepared:

Acid-hydrolyzed soybean meal gm 47 Acid-hydrolyzed corn meal gm 23Choice white grease ml 50 Non-ionic detergent (a tertiary octylphenoxyAutoclaved for 90 min. in 0.2 N H 80, and then adjusted to pH 6.5 withNaOH.

100 ml. aliquots of the medium were placed in a series of 500 ml.conical flasks which were then plugged, sterilized, and cooled. Eachflask was then inoculated with 4 ml. of each of a 48 hr. culture ofBla'keslea trispora NRRL 9216 and Blakeslea trispora NRRL 9159, andagitated on a Gump rotary shaker at 200 rpm. at 29 C. To experimentalflasks at Zero hrs, 24 hrs., or 48 hrs. respectively, 5 ml. of sterilekerosene was added and shaking resumed. Also at 48 hours 0.1 ml. ofsterile B-ionone was added to all flasks. Shaker incubation wascontinued to a total fermentation time of 144 hours before individuallyharvesting the mycelia from each flask by filtration 3,025,221 PatentedMar. 13, 1962 "ice followed by drying in a vacuum oven at 55 C. Theindividual yields were then ground in a Wiley mill, extracted withpetroleum ether (B.P. 33 C. to '57" C.), and the extracts comparedspectrophotometrically with an authentic sample of beta-carotene in thesame solvent, the results being shown in Table I.

EXAMPLE 2 The experiment of Example 1 was repeated with the exceptionthat the commercial kerosene hydrocarbon solvent of Example 1 wassubstituted by a commercial kerosene-type petroleum fraction hydrocarbonsolvent characterized by an initial B.P. of 386 R, an end B.P. of 484 E,a sp. gr. of O.7750.788 at 60 F., an API gravity of 49.8, a refnactiveindex at 20 C. of 1.4334, a flash point (closed cup at 0 F.) of /145, akauri butanol number of 29, an aniline pt. of 175 F., a pour point of-25 F and a zero iodine number). This results with this solvent areshown in Table II.

Table II Percent of specific commercial Time added Micrograms B-lraction added (hrs) carotene per 100 ml. culture Control (none) 48, 9085 added at start of fermentation (zero hrs.) 81, 736 5 48 74, 290

EXAMPLE 3 The experiment of Example 1 was repeated with the exceptionthat the kerosene ofEx-ample l was substituted by a commercialkerosene-type petroleum fraction characterized by an initial B.P. of 383F, and end B.P. of 415 F., a specific gravity at 60 F. of 0.7852, an APIgravity of 48.7, a refractive index of 1.4322 at 20 C., a flash point(closed cup at 0 F.) of 160 F., a kauri butanol number of 30, an anilinepoint of 169 F., and an iodine number of 6.5). The results are set forthin The experiment of Example 1 was repeated with the exception that thekerosene was substituted by a commercial kerosene-type hydrocarbonsolvent characterized by an initial B.P. of 376 R, an end B.P. of 468F., a sp. gr. at 60 F. of 0.7896, an API gr. of 47.7 a refractive indexat 20 C. of 1.4360, a flash point (closed cup) of F, a kauri butanolnumber of 31, an aniline point of 161 F., and an iodine number of 10.The results are set forth in Table IV.

EXAMPLE In this experiment flasks containing 150 ml. aliquots of afermentation medium corresponding to that of Example 1 but alsocontaining 5 percent kerosene were respectively inoculated with 4 ml. ofa 6-day culture of Blnkeslerz trispom NRRL 9159 or NRRL 9216. The flaskswere incubated at 28 C. on a Gump shaker rotating at 200 rpm. for 48hours, and then a fiask containing a kerosene-exposed culture ofBlakeslea trispora NRRL 9159 was combined with one containing akerosene-exposed culture of NRRL 9216. Then ml. aliquots of the combinedculture were used to inoculate fresh flasks of the fermentation mediumof Example 2 containing 5 percent of the specific solvent. Under thesame fermentation conditions as in Example 2, the results presented inTable V were obtained, thus showing that even better yields are obtainedif stock sub-cultures are exposed to a kerosene-type material.

The experiment of Example 2 was repeated with the exception that theconcentration of choice white grease was varied between 3 percent and 8percent. The results appear in Table VI, and show that the solvent andthe white grease are not functional equivalents.

Having disclosed our invention, we claim:

1. In the method of producing [El-carotene by fermenting on organismfrom the group consisting of Blakeslea trispora NRRL 9159, Blakesleatrispora NRRL 9216, and mixtures thereof in a tap water fermentationmedium comprising neutralized cereal grain hydrolysate, white grease, anonionic detergent, thiamine, and fl-ionone, the improvement comprisingthe steps of adding about 5% by volume (based on the fermentationmedium) of a petroleum hydrocarbon fraction at up to about 48 hoursafter beginning the fermentation, said petroleum hydrocarbon fractionbeing selected from the group consisting of kerosene and kerosenefractions characterized by having an initial B.P. of 376-386" R, an endB.P. of 415- 484 F., a sp. gr. at F. of 0775-0789, an API gravity of47.7-49.8, a refractive index at 20 C. of 1.4322- 1.4360, a closed cupflash point of -150 F., a kauri butanol number of 29-31, an anilinepoint of 161-175 F., and an iodine number of 0-10, and continuing thefermentation to a total period of about 144 hours.

2. In the method of producing fl-carotene by fermenting an organismselected from the group consisting of Blakeslea trispora NRRL 9159,Blakeslea trispora NRRL 9216, and mixtures thereof in an aqueousnutrient medium, the improvement comprising adding about 5% by volume(based on the fermentation medium) of a petroleum hydrocarbon fractionto said medium during fermentation, said hydrocarbon fraction being amember of the group consisting of kerosene and kerosene fractionscharacterized by having an initial B.P. of 376-286 F., and end B.P. of415-484 F., a sp. gr. at 60 F. of 0.775- 0789, an API gravity of47.7-49.8, a refractive index at 20 C. of 14322-14360, a closed cupflash point of 135-160 F., a kauri butanol number of 29-31, an anilinepoint of 161-175" F., and an iodine number of 0-10.

No reference cited.

1. IN THE METHOD OF PRODUCING B-CAROTENE BY FERMENTING ON ORGANISM FROMTHE GROUP CONSISTING OF BLAKESLEA TRISPORA NRRL 9159, BLAKESLEA TRISPORANRRL 9216, AND MIXTURES THEREOF IN A TAP WATER FERMENTATION MEDIUMCOMPRISING NEUTRALIZED CEREAL GRAIN HYDROLYSTATE, WHITE GREASE, ANONIONIC DETERGENT, THIAMINE, AND B-IONONE, THE IMPROVEMENT COMRPISINGTHE STEPS OF ADDING ABOUT 5% BY VOLUME (BASED ON THE FERMENTAION MEDIUM)OF A PETROLEUM HYDROCARBON FRACTION AT UP TO ABOUT 48 HOURS AFTERBEGINNING THE FERMENTATION, SAID PETROLEUM HYDROCARBON FRACTION BEINGSELECTED FROM THE GROUP CONSISTING OF KEROSENE AND KEROSENE FRACTIONSCHARACTERIZED BY HAVING AN INITIAL B.P. OF 376-386* F., AND END B.P. OF415484* F., A SP. GR. AT 60* F. OF 0.775-0.789, AND API GRAVITY OF47.7-49.8, A REFRACTIVE INDEX AT 50* C. OF 1,43221,4360, A CLOSED CUPFLASH POINT OF 135-150* F., A KAURI BUTANOL NUMBER OF 29-31, AN ANILINEPOINT OF 161-175* F., AND IODINE NUMBER OF 0-10, AND CONTINUING THEFERMENTATION TO A TOTAL PERIOD OF ABOUT 144 HOURS.